AIM 1- Solving Fec Signaling complex with FecA and periplasmic portions of FecR and TonB. [unreadable] X-ray crystal structures of FecA with FecR and TonB or soluble portions of these portions will illustrate the interactions of these proteins and how they are involved in the iron-citrate gene induction pathway. FecA has been solved in both an apo and a liganded form and diffracts up to 2.0 resolution (Ferguson et al., 2002; Yue et al., 2003). Starting with this structure and adding soluble portions of the other proteins is an understandable place to begin. The C-terminal (periplasmic) portion of FecR (FecR238-317) is sufficient to interact with FecA (Enz et al., 2003). The C-terminal portion of TonB is soluble and retains affinity for the TonB box as recently shown in crystal structures of FhuA and BtuB with TonB133-239 and TonB147-239 (Pawelek et al., 2006; Shultis et al., 2006). In these structures, the TonB fragment (not having its alpha helix which spans the inner membrane) will be unenergized. Therefore it would be a static picture of the complex in one potential transient state. However, studies of these iron-citrate bound and apo-FecA-TonB(C-ter)-FecR(C-ter) complexes would still indicate important interactions between all of these proteins and illustrate some portion of the signaling pathway. We have created a FecA construct with a cleavable N-terminal His-tag as well as N-terminal truncations FecA1-96, FecA1-86 and FecA1-74. We also have a periplasmic TonB fragment. We have cloned and expressed a C-terminal domain of FecR, FecR182-317, which aggregates in inclusion bodies as do all other FecR C-terminal truncations expressed. We created a coexpression construct with the FecA1-96 fragment to pursue soluble coexpression of this fragment. Refolding of FecR FecR182-317 is also being pursued.[unreadable] [unreadable] AIM 2- Solving the activated sigma factor complex FecIR1-85. [unreadable] The cytoplasmic portion of FecR, FecR1-85, constitutively increases the transcription of FecA and increases the affinity of the sigma factor FecI for the &#946;-subunit of RNA polymerase (Mahren and Braun, 2003). Our intent is to crystallize the FecIR1-85 with a truncation of &#946;-subunit of RNA polymerase, &#946;1-313. We intend to compare this to the heat stress related ECF sigma factor &#963;E in complex with its anti-sigma factor RseA. We have expression constructs of all of these constructs, however FecI expresses in inclusion bodies. We are pursuing both refolding and coexpression systems in order to achieve the purified complex of FecIR1-85 with a truncation of &#946;1-313. During the past year we have produced crystals of FecR1-85 which diffract X-rays to 2 A resolution. The protein packs with many copies in the asymmetric unit of the crystal, making structure solution difficult. Currently we are working on obtaining other crystal forms which are more amenable to structure solution, and we are also working on obtaining complexes between FecR1-85 and FecI.[unreadable] [unreadable] AIM 3- Pursuing the structure of the periplasmic iron-citrate binding protein FecB. Although FecB is not directly related to the signaling mechanism of the FecARI system it plays an important role in the transport of iron from FecA to the ABC transporter complex of consisting of FecC, FecD and FecE. It is not clear if the substrate of FecB is iron alone or iron-citrate as there is some evidence that iron is transported alone into the cytoplasm by the FecCDE transporter. Initial affinity studies by isothermal titration calorimetry indicate that iron-citrate is the substrate of FecB, however the affinities were too high to provide conclusive measurements. Extremely pure protein has been obtainable however crystallization has not yet proven fruitful so new constructs and purification conditions are being pursued. [unreadable] [unreadable] References[unreadable] Brooks, B.E. and Buchanan, S.K. (2007) Signaling mechanisms for activation of extracytoplasmic function (ECF) sigma factors. Biochim Biophys Acta, in press.[unreadable] Enz, S., Brand, H., Orellana, C., Mahren, S. and Braun, V. (2003) Sites of interaction between the FecA and FecR signal transduction proteins of ferric citrate transport in Escherichia coli K-12. J Bacteriol, 185, 3745-3752.[unreadable] Ferguson, A.D., Chakraborty, R., Smith, B.S., Esser, L., van der Helm, D. and Deisenhofer, J. (2002) Structural basis of gating by the outer membrane transporter FecA. Science, 295, 1715-1719.[unreadable] Mahren, S. and Braun, V. (2003) The FecI extracytoplasmic-function sigma factor of Escherichia coli interacts with the beta' subunit of RNA polymerase. J Bacteriol, 185, 1796-1802.[unreadable] Pawelek, P.D., Croteau, N., Ng-Thow-Hing, C., Khursigara, C.M., Moiseeva, N., Allaire, M. and Coulton, J.W. (2006) Structure of TonB in complex with FhuA, E. coli outer membrane receptor. Science, 312, 1399-1402.[unreadable] Shultis, D.D., Purdy, M.D., Banchs, C.N. and Wiener, M.C. (2006) Outer membrane active transport: structure of the BtuB:TonB complex. Science, 312, 1396-1399.[unreadable] Yue, W.W., Grizot, S. and Buchanan, S.K. (2003) Structural evidence for iron-free citrate and ferric citrate binding to the TonB-dependent outer membrane transporter FecA. J Mol Biol, 332, 353-368.